This project is designed to provide new information on the biochemical basis for virulence in Mycoplasma pneumoniae infections, particularly primary atypical pneumonia (PAP) of humans. This phase of our long-term study is concentrating on the nature of the receptor site to which the mycoplasma attaches. We have already established the fact that the receptor site on lung fibroblasts is a sialoglycoprotein in the 100,000 dalton range. Recent data indicate that glucosamine is a major carbohydrate in this molecule. We next will isolate the M. pneumoniae receptor by using lectin affinity chromatography. Crude receptor prepared by triton X-100 solubilization of lung fibroblast membranes will be added to wheat germ agglutinin/sepharose, and glycoproteins will be eluted with N-acetyl glucosamine. Purified receptor will be collected after ultrafiltration and concentration, and characterized by polyacrylamide gel electrophoresis and chemical assays for protein and carbohydrate. Specificity and activity of purified receptor glycoprotein will be determined by the degree of blocking of M. pneumoniae attachment to lung fibroblast cells in vitro. The glycoprotein receptor will be used as an antigen to prepare monoclonal antibodies in mouse-mouse hybridomas. Specificity of immunoglobulins and stability of clones will be assessed by ELISA techniques with a horseradish peroxidase label. The availability of purified receptor and its homologous antibody will permit us to determine the number and distribution of receptor sites on host cell membranes. The receptor molecules will be covalently coupled to Sepharose to develop an improved isolation and recovery method for the P1 protein, shown by several laboratories to be equivalent to the active binding site of the M. pneumoniae organism and a prime candidate for vaccine antigen. We also will determine the frequency of occurrance for the M. pneumoniae receptor site on various cell types, and correlate it with the ability of M. pneumoniae to attach to those cells. Conversely, we will examine the ability of other mycoplasmas to use this receptor as an attachment site. Our ability to isolate and study the M. pneumoniae receptor, along with its specific antibody, will provide a unique perspective on the molecular basis of pathogenicity, while being of direct value to developing effective therapy and prophylaxis for mycoplasma disease.